The concentrations of 5-HT in serum and colon were increased in LPS-exposed offspring. Meanwhile, the expression level of tryptophan hydroxylase 1 (TPH1) in the colon was increased after maternal LPS treatment, whereas SERT was reduced. Furthermore, Golgi-Cox staining showed that neuronal dendritic length and spine density were significantly reduced in the mPFC of LPS-exposed offspring. The current study reveals that maternal LPS treatment resulted in an exaltation of the 5-HT of mPFC in ASD-like young rats, which may partly be caused by the abnormal elevation of 5-HT metabolism in its colon.Autism spectrum disorder (ASD) is a neurodevelopmental disorder characterized by reduced social interactions, impaired communication, and stereotyped behavior. The aim of this research is to investigate the changes in serotonin (5-HT) in the medial prefrontal cortex (PFC) of autism-like offspring induced by maternal lipopolysaccharide (LPS) exposure. Pregnant Sprague-Dawley rats were intraperitoneally injected with LPS to establish an autism-like model in their offspring. Offspring prenatally exposed to LPS showed autism-like behavior. The serotonin level in the mPFC of 2-week-old offspring was noticeably increased after maternal LPS exposure. Differentially expressed genes (DEGs) were enriched in pathways related to tryptophan metabolism and the serotonin system, as shown in RNA-seq findings. Consistently, tryptophan and serotonin metabolisms were altered in 2-week-old LPS-exposed offspring. The mRNA expression levels of 5-HT catabolic enzymes were remarkably reduced or tended to decrease. Moreover, maternal LPS exposure resulted in a higher serotonin 1B receptor (5-HT Autism spectrum disorder (ASD) is a group of neurodevelopmental disorders characterized by various behavioral symptoms such as social deficits, repetitive behaviors, and anxiety . To a grAs a key neurotransmitter, 5-HT regulates multiple aspects of neural development ,8. AbnorTo investigate this hypothesis, the rats were first treated with LPS during pregnancy to establish an ASD-like offspring model, and the 5-HT level of the mPFC was measured. Then, we combined RNA-seq techniques and tryptophan target metabolomics to identify abnormalities in 5-HT metabolism-related signaling pathways of the mPFC in the offspring. Finally, the expression levels of metabolic enzymes and 5-HTRs were confirmed in both the mPFC and colon of LPS-exposed offspring. The current study offers an experimental foundation for the possible mechanism of 5-HT alterations during the early neurodevelopment period.Sprague-Dawley (SD) rats were acquired from the Animal Experiment Centre of Chongqing Medical University  and raised in a specific pathogen-free environment with a 12 h light-dark cycle  at a constant temperature (22–24 °C). The animal use protocol has been reviewed and approved by the Ethics Committee of the Children’s Hospital of Chongqing Medical University .n = 40 from 6 litters of rats) and PBS (n = 40 from 6 litters of rats) groups, respectively.Eight-week-old female rats were paired with two male rats of the same strain and maintained overnight. Then, female rats with a vaginal plug were observed the next morning. These females were observed at 0.5 days of gestation. Meanwhile, they were randomized to the PBS and LPS groups. LPS  was dissolved at 50 µg/mL in phosphate-buffered solution (PBS) and administered intraperitoneally to the pregnant rats in the LPS group on 9.5 days of gestation (100 µg /kg) . Similarn = 20 per group) during the day (light-on period). The tests were conducted in a peaceful and mild environment to ensure the quality of the experiment. Before the next set of rats were tested, the test boxes and cages were cleaned of urine and feces and then disinfected with 75% alcohol. All behavioral tests were performed with 6- to 8-week-old rats (The open-field test was conducted according to a previous methodology . The rat2) with three chambers (20 × 40 cm2) and entrances (6 × 6 cm), allowing access to the side chambers. The test consisted of two 5 min stages: the social interaction test and the social novelty test. The social interaction test involves an age- and sex-matched stranger rat and a tiny item in the two side chambers [The three-chamber sociability test was conducted in a white plexiglass box  was first quickly frozen in liquid nitrogen and then frozen for long-term preservation at −80 °C. Meanwhile, the serum and colon of each subject were extracted and stored at −80 °C. The tissue collection was completed by two experiments that were blinded to the group assignments.n = 10 per group) and colon  was rinsed away, and the tissue was homogenized in 1× PBS (100 mg tissue/1 mL PBS). The tissue was ground into pieces, and the homogenates were centrifuged at 5000× g for 5 min at 4 ℃. The supernatant  was collected, and the 5-HT concentration was determined using an ELISA kit , as directed by the manufacturer. The absorbance was measured at 450 nm, and the optical density values were computed in triplicate using a standard curve.Residual blood from the mPFC (n = 4 per group) were perfused transcardially and sacrificed at 2 weeks of age, and their brains were totally exfoliated and preserved in 4% paraformaldehyde. Then, the brain was dehydrated with 4% paraformaldehyde containing 15% and 30% sucrose in that order. The brain was then sectioned into 15 μm thick sagittal slices by using a cryostat  after the tissue had sunk to the bottom. Then, 0.3% Triton X-100 was applied to the frozen slices for 20 min before blocking them with 5% BSA  for 1 h at room temperature. The slices were then incubated at 4 °C overnight with primary antibodies, including 5-HT  and 5-HT1BR . Fluorescent secondary antibodies, including donkey anti-rabbit  and donkey anti-mouse , were applied at room temperature in the dark for 1 h. Nuclei were labeled with DAPI solution . Finally, fluorescent images of the mPFC were captured using a Nikon automated bioluminescence microscope at a magnification of 400×. A single color channel analysis was performed using Image J software (version 1.8.0) to quantify the mean gray value. Mean gray value = Integrated Density/Area.The rats (n = 4 per group) was immersed in a 10 mL mixed solution and stored at room temperature in the dark for 2 weeks. Two weeks later, the tissue was transplanted into the same volume of solution 3 and maintained for 3 days at 4 °C. The brain was cut at the coronal plane (200 µm thick), and the Hito Golgi-Cox OptimStain PreKit  was used for section staining. ImageJ software and NeuronStudio software (version 0.9.92) were used for analyzing the dendritic length and spine density, respectively.Golgi-con staining was performed using the Hito Golgi-Cox OptimStainTM Kit  One day before staining, solution 1 and solution 2 were mixed evenly in a 1:1 ratio and stored at room temperature in the dark. After careful extraction and washing in PBS, the brain  was used as the reference gene for all target genes. As instructed by the manufacturer, the Total RNA Extraction Kit  was used for RNA extraction from the samples. The RNA concentration was measured using the NanoDrop ONE System . The PrimeScript RT Reagent Kit  was used for cDNA synthesis. The quantitative polymerase chain reaction was performed using SYBR Green Mix  on a real-time PCR system  for 40 cycles . The relative expression of the target genes was calculated using the 2n = 4 per group) and colon (n = 4 per group). Protein concentrations were detected using the BCA Protein Assay Kit  and a microtiter plate reader . For Western blotting, the proteins were separated through sodium dodecyl sulfate-polyacrylamide gel electrophoresis and transferred onto 0.45 µm polyvinylidene difluoride membranes . All membranes were incubated with primary antibodies against TPH1 , TPH2 , SERT , 5-HT1BR , and GAPDH  at 4 °C overnight, followed by 1 h incubation at room temperature with secondary antibodies. The image was photographed using a Syngene GBox Imaging System  and an enhanced chemiluminescence solution . Lastly, the expression level of the target protein bands was measured using Image Lab (version 3.0) software.RIPA lysis buffer  and 10% protease inhibitor cocktail  were used for total protein extraction of the mPFC (n = 10 per group). The sequencing experiment was conducted using the Illumina TruseqTM RNA sample prep kit, according to the manufacturer’s protocol. Total RNA was extracted from the tissue samples. The concentration and purity of the proposed RNA were examined using Nanodrop2000 . Then, mRNA was enriched using Oligo(dT) beads. With the addition of fragmentation buffer, mRNA broke, and small fragments of approximately 300 bp were isolated using magnetic beads. In the presence of reverse transcriptase, first-strand cDNA was synthesized using mRNA as a template, which was followed by second-strand synthesis. Then, End Repair Mix and poly (A) were added to the cDNA fragments. Finally, mRNA sequencing was performed at Majorbio Company  using the Illumina Novaseq 6000 sequencing platform . DESeq2 was used to identify differentially expressed genes (DEGs) with a fold change of >1 and p < 0.05. To investigate the DEG functions, Gene Ontology (GO), Kyoto Encyclopedia of Genes and Genomes (KEGG), and Reactome enrichment analyses were executed. The mPFCs of the PBS and LPS groups were subjected to RNA sequencing (n = 10) and LPS (n = 10) offspring were sent for tryptophan target metabolomics. Each group contained two replicates. For further testing, a 50 mg aliquot of each individual sample was carefully weighed. After 500 μL of extract solution  was added, the samples were vortexed for 30 s, homogenized at 35 Hz for 4 min, and sonicated for 5 min in an ice-water bath. The homogenate and sonicate circles were performed twice before subsiding for 1 h at −40 ℃. Following centrifugation , a 320 μL aliquot of the supernatant was transferred to an Eppendorf tube. The supernatant was then dried under a moderate nitrogen stream before being reconstituted in 80 μL of water containing 0.1% formic acid. The clear supernatant was used for UHPLC-MS/MS analysis after centrifugation . The UHPLC separation was performed using an EXIONLC System (Sciex) outfitted with a Waters ACQUITY UPLC HSS T3 column . For assay development, a SCIEX 6500 QTRAP+ triple quadrupole mass spectrometer (Sciex) with an IonDrive Turbo V electrospray ionization interface was used. SCIEX Analyst Work Station Software (Version 1.6.3) and Sciex MultiQuant software (Version 3.0.3) were used to collect and process data.The mPFCs of the PBS (t-test and Graphpad Prism software (version 9.0). p < 0.05 was considered statistically significant.The quantitative data were presented as mean ± standard deviation (SD). Between-group comparisons were performed using the Student’s t38 = 3.222, p < 0.05). However, no noticeable difference was observed in total traveled distance between the PBS and LPS groups . In addition, the rats in the LPS group spent more time grooming themselves than those in the PBS group . These data revealed that maternal LPS exposure reduced the ability to explore novel environments but increased repetitive behaviors and did not impair the locomotor activity of offspring. To assess if prenatal exposure to LPS causes ASD-like behaviors in offspring, we performed behavioral tests such as the open-field and three-chamber sociability tests. Using the open-field test, the autonomic inquiry activities of the rats in an unfamiliar environment were measured. After analyzing the locomotor activity, line crossing in the LPS group was found to be less than that in the PBS group  and less time in the stranger rat chamber  than the PBS offspring. Additionally, t38 = 2.938, p < 0.01) and less time in the stranger rat chamber . These findings suggest that maternal LPS exposure impairs social interaction and social novelty recognition in its offspring.A three-chamber sociability test can be used to evaluate the social behavior of rats and determine whether it is abnormal. To characterize alterations in gene expression triggered by gestational LPS exposure, RNA-seq was performed using the mPFC of the PBS and LPS groups. In total, 287 DEGs were discovered. Of these DEGs, 170 were upregulated and 117 were downregulated A. The voGO, KEGG, and Reactome pathway enrichment studies were conducted to gain a deeper understanding of the identified DEGs. The GO analysis and KEGG analysis suggested that DEGs were enriched in synaptic-related GO terms and tryptophan metabolism pathways . Reactomt18 = 3.065, p < 0.001). t10 = 3.472, p < 0.01). Thus, LPS exposure during the pregnancy period induces the serotonin system in the mPFC of the LPS offspring.The 5-HT content of the mPFC of 2-week-old offspring was determined using the enzyme-linked immunosorbent assay and immunofluorescence staining to explore whether LPS treatment during gestation causes any change in the serotonin system in the mPFC. As demonstrated in t17 = 3.220, p < 0.01). The 5-HT level in the serotonin pathway exhibited a slightly rising trend in the LPS group, although no significant difference was observed between the LPS and PBS groups. However, the amount of 5-hydroxyindoleacetic acid (5-HIAA), a downstream 5-HT metabolite, was significantly lower in the LPS group than in the PBS group . Furthermore, in the kynurenine pathway, 3-hydroxykynurenine (3-HK) concentration was obviously lower in the LPS group than in the PBS group . Whereas no distinct differences in the indole pathway were observed between the LPS and PBS groups . We then detected the mRNA expression levels of 5-HT catabolic enzymes, including monoamine oxidase type A (MAOA), arylalkylamine N-acetyltransferase (AANAT), and acetylserotonin O-methyltransferase (ASMT). The findings demonstrated that the LPS-exposed offspring exhibited considerably lower MAOA mRNA expression levels . Meanwhile, AANAT and ASMT trended toward lower mRNA expression levels . According to the aforementioned findings, LPS therapy during pregnancy may affect tryptophan metabolism, especially the kynurenine and serotonin pathways, in the mPFC of the offspring.Tryptophan metabolism occurs through the kynurenine or serotonin pathway and produces bioactive metabolites . Gut mic1BR has been linked to social and communicative activities [1BR) protein expression in the mPFC of the offspring were detected by Western blotting and immunofluorescence. According to the quantitative analysis of qPCR and Western blot, the mRNA and protein expression levels of TPH2 and SERT exhibited no significant differences between the LPS and PBS groups . The 5-HT1BR mRNA and protein expression levels were significantly increased in the LPS offspring compared with the PBS offspring . Using immunofluorescence staining, we observed that the 5-HT1BR levels were noticeably higher in the LPS group than in the PBS group , which is consistent with the qPCR and Western blot results. These results reveal that maternal LPS therapy affects serotonin biological function through the expression of its receptor 5-HT1BR in the mPFC, whereas it does not impact 5-HT synthesis and reabsorption in the mPFC. Neurons were morphologically observed using Golgi-Cox staining in the mPFC of 2-week-old offspring to further investigate the association between the change in the 5-HT level and ASD-like behavior resulting from maternal LPS exposure. The dendritic branches of the neurons were noticeably shorter in the LPS group than in the PBS group . Meanwhile, a remarkable reduction in spine density was observed in the LPS offspring . Together, the present findings confirm that maternal LPS exposure contributes to the abnormal morphology of dendrites during the early postnatal period. Tryptophan hydroxylase (TPH) is the rate-limiting enzyme of the 5-HT synthesis pathway, including TPH1 and TPH2, which are responsible for 5-HT synthesis of the intestinal and central nervous systems, respectively ,20. 5-HTtivities ,23. Thentivities . To deteBS group F. Accordt22 = 4.463, p < 0.001). t18 = 2.680, p < 0.05), also consistent with the alteration in the mPFC. Furthermore, the mRNA and protein expression levels of both TPH1 and SERT displayed notable elevation and reduction, respectively, in the colon of LPS offspring at 2 weeks compared with the PBS offspring . To sum up, these are crucial findings related to the understanding of the increased 5-HT level in the mPFC, and we speculated that the changes in 5-HT metabolism in the colon may have a certain effect on the 5-HT level in the mPFC during the period of early-life neurodevelopment.Considering that 90% of 5-HT is synthesized in the intestine, 5-HT levels as well as TPH1 and SERT protein expression were assessed using Western blotting and immunofluorescence. Immune system activation during pregnancy increases the risk of neurodevelopmental disorders in the offspring. The method of constructing ASD animal models through maternal LPS exposure is well established ,26. ConsThe association between ASD and 5-HT system dysfunction has been known for a long time ,28. PrevTph2−/− mice have ASD-related behavioral consequences [Sert−/− mice also exhibited ASD-associated behavioral changes [1BR [1BR agonist challenge induces autism-related behavior in mice [1BR in the mPFC of 2-week-old offspring. These results imply that LPS had no effect on 5-HT synthesis or degradation but did affect the expression of the receptor in the mPFC during the early neurodevelopment period.Many key proteins are involved in the anabolism, catabolism, and function of 5-HT. Most reports have supported the conclusion that TPH2, the initial and rate-limiting enzyme in 5HT production, is related to ASD ,39. For equences ,41. SERTequences . By servequences . A previequences . At the  changes . However changes . Among tges [1BR ,23. Laws in mice . AccordiNumerous studies have elucidated that the serotonin system is involved in the growth of neuronal systems ,47. Besi5-HT cannot cross the BBB. Nonetheless, the BBB does not fully mature until tight junction-specific proteins appear in rats at approximately postnatal day 20 . MoreoveAlthough our study made some important discoveries, there are limitations. First, multiple comparisons were not conducted in the current study. Second, a major experiment is urgently required to support the colon-central nervous system crosstalk to regulate 5-HT metabolism. Finally, further research is also required to fully comprehend the mechanisms through which the 5-HT system regulates nervous system development. Overall, our work clarifies that maternal LPS exposure increases the 5-HT level in the mPFC of the young offspring, which may be attributable to the ASD-related behavior in adult rats that is potentially induced by the high 5-HT level in the colon.


The dissolved oxygen could reach 14 mg/L after adding 80 mg/mL of ZD-CAR. Meanwhile, gold nanoparticles could convert light energy into heat energy under 808 NIR irradiation to induce local superheating and kill tumour cells. In summary, this study developed a nanoplatform that combines chemo-photothermal-targeted therapy. It has shown good therapeutic effeciency in cellular experiments and performance tests and has promising applications in anti-cancer therapy.The toxic effects of chemotherapy drugs on normal tissues are still a major limiting factor in cancer treatment. In this paper, we report a metal-organic framework (Zn-Co ZIF) with chitosan-coated outer layer as a carrier for the drug adriamycin hydrochloride (DOX), a treatment for liver cancer, as a novel anti-cancer nanodrug-enhanced carrier. Gold nanoparticles, a good photothermal conversion agent, were combined with the target SH-RGD during surface functionalisation to prepare Zn-Co ZIF@DOX-CS-Au-RGD (ZD-CAR), a nanoplatform with good photothermal conversion properties and targeting for combined liver cancer therapy. ZD-CAR was developed after RGD accurately targeted the tumour and entered the tumour microenvironment (TME), it cleaves and releases the liver cancer therapeutic agent (DOX) in a weak acidic environment to effectively kill tumour cells. The metal skeleton cleavage releases Co TradiSo far, metal-organic frameworks (MOFs) which consist of metal ions and organic ligands are frequently prescribed by many biomedical researchers as promising nanocarriers due to their adjustable size, very high specific surface area inside and outside their pores, better biocompatibility, and good pH responsiveness –16. HereLiver cancer is one of the most common types of cancers. DOX (Adriamycin) has shown good efficacy in the treatment of liver cancer, but it has poor therapeutic activity and side effects on normal tissues, such as hepatotoxicity . These lCompared to single treatment modalities, the combination of chemo-photothermal therapy , 31 is nIn general, stimulatory drug delivery systems , 43 requ2+, which can catalyze H2O2 to produce oxygen. Generation of O2 can alleviate the tumor hypoxic environment (In vitro experiments have demonstrated that Zn-Co ZIF@DOX-CS-Au-RGD (ZD-CAR) has good anti-tumor efficacy with no significant adverse effects , Zn-Co ZIF. As shown in ironment , 38. Meaironment . In orde effects –46. Ther22.14H6N2, 2-MIM), Doxorubidn hydrochloride (DOX), chitosan (CS), mercaptopropionic acid (MPA), zinc nitrate hexahydrate (Zn(NO3)2•6H2O), chloroauric acid, cobaltous nitrate hexahydrate (Co(NO3)2-6H2O), methanol, 1-Ethyl-(3-dimethylaminopropyl) carbodiimide hydrochloride (EDC), N-Hydroxysuccinimides (NHS), SH-RGD and sodium borohydride, hydrogen peroxide . Penicillin/Streptomycin Dual Antibody, Propidium iodide (PI), DMEM, Calcein-AM, DMSO and Fetal calf serum and Trypsin.The following materials were used for the study: 2-Methylimidazole (C2.23)2·6H2O was dissolved in 100 mL of methanol and mixed well. 1.620 g of 2-MIM was dissolved in 100 mL of methanol and mixed well. Then, the mixture of the metal ion was poured into the 2-MIM solution and stirred for 12 h. It was washed with methanol and centrifuged. Then, it was dried to obtain Zn-Co ZIF product  system. Subsequently, the successful encapsulation of DOX was verified by testing the reduction in BET (Brunner-Emmet-Teller). The load efficiency of DOX can be calculated by the following equation:1/C2 is the concentration of the supernatant after the first or second  propionic acid N-hydroxysuccinimide ester (SPDP) and stirred for 3 h. After they were centrifuged, 500 μL of the prepared Au NPs was added to the ZD-C solution. It was stirred smoothly to attach the Au NPs to the outside of the ZD-C. Then, ZD-CA nanoparticles were obtained.2.7The target products ZD-CAR nanoparticles were obtained by attaching mercaptopropionic acid and SH-RGD to the surface of ZD-CA nanoparticles through the action of Au-S covalent bonds. This was done by adding 2 mL RGD (3.4 mg/mL) and 500 μL mercaptopropionic acid. It was stirred for 5 h and centrifuged. Then, ZD-CAR was obtained.2.8ZD-CAR nanoparticles were characterized by SEM and TEM in 2.9To evaluate the photothermal ability of ZD-CAR nanoparticles, the dispersions of aqueous solutions were irradiated under 808 nm NIR with different concentrations to observe the change in temperature with time; while their photostability was tested by repeatedly increasing and decreasing the temperature and withdrawing the laser after ten minutes of continuous illumination .2.104 mg of ZD-CAR was dissolved in PBS at pH 7.4, 6.8 and 5.7. It was shaken in a constant temperature shaker at 37°C, and centrifuged at different time points. The same volume of supernatant was added to the same volume of PBS, and the wavelength of UV emission was measured at different times.2.112O2, and the O2 produced was tested using a JPB-607A dissolved oxygen instrument.Different concentrations of Zn-Co ZIF and ZD-CAR nanoparticles were added to an aqueous solution containing the same concentration of H2.122 cells were cultured in a medium supplemented with 10% fetal bovine serum (FBS) and 1% double antibodies (penicillin/streptomycin). Then, they were incubated in a 5% CO2 incubator at 37°C. The medium was changed every day. The cells were isolated with trypsin-EDTA, and they were passaged and seeded.HepG2.13in vitro was tested using HepG2 cells by CCK-8 reagent. A certain number of cells were inoculated in 96-well plates and incubated overnight in a 5% CO2 incubator at 37°C, after which the cells were washed with PBS. The same concentrations  of the different types of nanoparticles were placed in the medium and the cells were incubated. Three replicate wells were set up for each concentration. After 24 h incubation, the effect of the different concentrations of the drug on cell viability was measured using the CCK-8 kit. The absorbance of the well plates at 450 nm was measured using an enzyme marker, and cell viability was calculated. To test the effect of light duration on cell viability, PBS-treated cells and cells treated with 100 μg/mL ZD-CAR drug were used as controls and irradiated at different times. Then, the plates were stained using CCK-8 and the absorbance of the well plates at 450 nm was measured using the Tecan Sunrise. The following formula was used to calculate its cell viability:The cytotoxicity of ZD-CAR control represents the absorbance of untreated cells and ODblank means the culture without samples or cells and ODsample indicates the cells with different treatments (where ODeatments .2.142 cells were cultured under normoxic and hypoxic conditions respectively. The cells cultured under different conditions were stained with hypoxia-inducible factor (HIF-1α) and their fluorescence intensity was observed under a fluorescence microscope, after which the cells were incubated in normoxic and hypoxic environment using different materials, and cell viability was obtained after 24h of incubation using the CCK-8 kit to measure the absorbance of the well plates at 450nm using an enzyme marker.HepG2.152 cells were incubated overnight for wall attachment using confocal dishes. Then, they were treated with different types or concentrations of material and stained with different dyes. Subsequently, they were observed under confocal laser scanning microscopy (CLSM) or fluorescence microscopy.HepG2.162 cells were inoculated into 6-hole plates at a density of 3×105 per well and incubated overnight. The medium was aspirated and a fresh medium containing 200 μg/mL concentration of DOX, ZIF, and ZD-CAR, respectively was added. It was divided into six groups: addition of PBS with light only, addition of DOX, addition of ZIF, addition of ZD-CAR, and addition of ZD-CAR with light treatment. After it was incubated with drugs for 6 hours, the medium was discarded, and washed twice with PBS. Another fresh medium was added. The light only and ZD-CAR + light groups were exposed to 808 laser and power of 2 W·cm-2 for 5 minutes. Then, the cells were digested using EDTA-free trypsin. They were collected, washed, and centrifuged. The cells were treated with Annexin V-FITC/PI staining according to the manufacturer’s instructions. They were incubated for ten minutes at room temperature and protected from light. Then, they were analyzed using a flow cytometer on the machine.Apoptosis assay of hepatocellular carcinoma cells was performed using Annexin V-FITC/PI double staining apoptosis assay kit. HepG2.17in vitro results, we established a HepG2 mouse model to determine the role of nanoparticles such as ZD-CAR in the treatment of tumours in mice in vivo. All experimental female mice  were purchased from Guangdong Viton Lihua Laboratory Animal Technology Co., Ltd. Firstly, 1×106 HepG2 cells were injected subcutaneously under the axilla of each mouse to establish the tumour model. Then, the mice were divided into 7 groups (5 mice per group) according to the materials, grouped as: PBS, Light, Zn-Co ZIF, ZD, DOX, ZD-CAR and ZD-CAR+L. The PBS group was treated with neutral PBS only as a control group, and the Light group was used as a pure light control group. For treatment, treatment was started when the tumour size reached about 120 mm3 and the treatment date was day 0. The same dose of drug was injected into the mice by tail vein injection for treatment and data such as the length and width of the tumour and its body weight were recorded for each mouse in each group every three days and the volume of the tumour (V) could be calculated by the formula. The formula is: V=0.5×(length)×(width)2.With the expected 33.1As shown in As illustrated in 3.2The results in 3.32O2 was added, and the dissolved oxygen of both materials rises with increase in their concentration. With the addition of 80 μg/mL concentration and at about 15 min of reaction, the dissolved oxygen of bare Zn-Co ZIF could reach up to 20 mg/L; while that of ZD-CAR could get to 14 mg/L. Oxygen is produced due to the presence of Zn-Co ZIF  laser and the solution temperature was monitored using thermocouple thermometer (OMEGA HH520). In  and hypoxic (1%) conditions. We know that DOX is more toxic in the presence of a certain amount of oxygen and has a better therapeutic effect. As shown in Hypoxia-inducible factor (HIF-1α) will upregulat under hypoxic conditions, so we used HIF-1α staining to detect the degree of hypoxia. As shown in 3.5.32 cells were treated with the same concentration of ZD-CAR nanomaterials at different incubation times. This was followed by PI staining and observation under confocal laser scanning microscopy (CLSM). It can be seen that a small number of cells started to die at around 30 min, and as the incubation time was extended, the cells reached a high mortality rate at 200 min. This shows that the material has good therapeutic efficacy.As shown in As shown in 2 cells diminished and began to show orange fluorescence. This demonstrates that the chemotherapy and photothermal treatment had an effect. However, after treating the cells simultaneously with ZD-CAR and 808 laser, almost all the cells showed green fluorescence. This shows that their mitochondrial function was severely affected by the combined treatment and could cause cell death with high efficiency. The ratio of fluorescence intensity was shown in As shown in 3.5.4As shown in 3.5.5PBS is a buffered solution that is commonly used as a solvent to disperse nanoparticles. The PBS group was used as a control group, the Light group to observe the effect of light alone on the growth of mice, and the Zn-Co ZIF group was set up to observe the effect of the metal-organic skeleton on the growth of mice. After 26 days of treatment and recording the data, Blood is an important component in maintaining the vital signs of animals. After execution of the mice, blood was obtained from the mice by means of eye blood sampling and routine blood tests were performed to obtain in vivo, indicating that it has good biocompatibility and biosafety.In order to investigate whether our Zn-Co ZIF@DOX-CS-Au-RGD has good biosafety, one mouse from each group was randomly selected for execution at the end of treatment, and the heart, liver, spleen, lungs and kidneys were sectioned and fixed with tissue fixative Paraformaldehyde (4%) was used for fixation, followed by histological analysis. The results of the experiments are shown in in vivo.Terminal deoxynucleotidyl transferase-mediated dUTP nickel-end labeling (TUNEL) staining further confirmed that the ZD-CAR and ZD-CAR+L groups had more apoptotic cell regions than the control group (42+ and Zn2+ was successfully prepared in this study. Using the pH-sensitive property of easy cleavage under acidic conditions, it can be smoothly cleaved to release Co2+ and DOX under the tumor microenvironment. Co2+ can catalyze H2O2 to produce O2 to alleviate the tumor hypoxic environment, while the DOX released can be used for chemotherapy. In the meantime, the attachment of gold nanoparticles can confer good photothermal sensitivity for photothermal therapy, and secondly, the SH-RGD attached to the surface of the nanoparticles has good tumor targeting, so it cannot harm healthy tissues. After the material was prepared, its various components were characterized and various tests were done for its performance. The in vitro material tests and experiments such as cytotoxicity and confocal confirm the good therapeutic effect of the ZD-CAR nano-drug delivery platform, which provides a new idea for the combined chemotherapy and photot hermal treatment.In summary, a MOF ZD-CAR material containing both CoThe original contributions presented in the study are included in the article/CY: Investigation, Data Curation, Writing – Original Draft. ST: Validation, Formal analysis, Writing – Review & Editing. LG: Methodology, Supervision. GA: Resources, Writing – Review & Editing. HZ: Data Curation, Writing – Review & Editing. JH: Writing – Review & Editing. LJ: Investigation. ZB: Formal analysis. YZ: Funding acquisition, Project administration. NW: Methodology, Conceptualization, Supervision. All authors contributed to the article and approved the submitted version.


Endometrial cancer (EC) is the sixth most common malignancy among females worldwide. Due to limited therapeutic options, treatment of advanced or recurrent disease is associated with poor outcomes. The aim of this study was to describe the real-world treatment of patients with advanced or recurrent EC who received a systemic treatment following platinum-based chemotherapy.This retrospective cohort study was based on anonymized German claims data covering the period between January 1, 2010, and June 30, 2020. Patients with EC who started an anticancer treatment following platinum-based chemotherapy were observed for a minimum follow-up of 12 months. Available claims data were used to describe patient characteristics, subsequent treatment lines, healthcare resource utilization, and overall survival (OS) of patients.Out of 713 patients with advanced or recurrent EC and who had received a platinum-based treatment, 201 (mean age: 68.9 years) with a post-platinum-based treatment were identified and observed. The median OS in this population was 335.0 days. Of the 201 patients, 79 patients (39.3%) received a second line of treatment (LOT), and 21 patients (10.4%) had 3 or more treatment lines. In the LOTs following platinum-based chemotherapy, more than 70 different treatment regimens were observed. The hospitalization rate was generally high, with 5.2 hospitalizations per patient-year in the follow-up period.The wide variety of therapeutic regimens applied in patients in Germany who progressed after platinum-based therapy confirms the lack of therapeutic strategy for these patients, and the poor prognosis highlights the urgent need for new treatment strategies.The online version contains supplementary material available at 10.1007/s00432-022-04183-y. Endometrial cancer (EC) is the sixth most common malignancy among women worldwide, with an estimated incidence of about 417,367 new cases globally  system  and overall survival (OS) of these patients.This retrospective cohort study was based on anonymized claims data covering the period from January 1, 2010, to June 30, 2020, provided by the German statutory health insurance fund AOK PLUS. This dataset covers approximately 3.4 million individuals from the German federal states of Saxony and Thuringia, representing around 4% of the German population.German claims data provide information on patients’ demographics  and detailed reimbursement claims on outpatient care, inpatient care, pharmaceutical treatments, therapeutic devices, rehabilitation, and sick leave.Outpatient care data comprise information on diagnostic and therapeutic procedures   or two confirmed outpatient EC diagnoses made by a specialist  were recorded between January 1, 2010, and June 30, 2019. The analysis sample considered identified prevalent patients with EC who received platinum-based therapy (identified via ATC code L01XA- or OPS 8–54) and then initiated a second regimen, and who did not receive any platinum-based treatment in the preceding 12 months (platinum-based treatment-free period); this period was chosen to identify patients with EC who received their first platinum-based treatment. Based on current treatment guidelines recommending chemotherapy only for patients with advanced disease, all patients who had received platinum-based therapy were considered to have advanced stage disease . The observation ended at the patient’s death, the end of insurance, or the end of data availability .A fixed 12-month pre-index period (baseline) was used to describe patient characteristics at the time of the post-platinum-based therapy. Additionally, a patient individual pre-index period covering the entire available period between the first-ever observed EC-related treatment start until the index date (modified baseline period) was considered in order to describe the treatment history.Besides age, patients’ baseline profiles were focused on comorbidity status. The burden of specific comorbidities and the presence of other malignancies were assessed based on confirmed outpatient diagnoses and inpatient diagnoses documented during the 12-month pre-index period. One diagnosis was sufficient to define a patient as having the comorbidity/disease. Based on the diseases identified, the overall comorbidity status was expressed by calculating the Charlson Comorbidity Index (CCI)  was described, based on inpatient and outpatient care and outpatient prescriptions.For all categorical variables, the number and percentage of patients in each category were reported. Summary statistics, including mean, standard deviation (SD), and range, were applied for all continuous variables.All-cause OS after the start of the first post-platinum-based treatment (index date) was assessed by Kaplan–Meier analysis. Failure was defined as the date of all-cause death, with observations censored when insurance coverage ended or the end of data availability  was reached. Based on Kaplan–Meier estimates, the median survival time and the proportion of patients who survived 3, 6, 12, and 24 months were reported.The real-world treatment pattern was described for the patient individual post-index period after the start of a post-platinum-based therapy.Hysterectomy, lymphadenectomy, and radiotherapy  were considered as EC-related nonpharmacological treatments  was defined as the first prescription or first inpatient treatment with an EC-related agent at least 3 months after the platinum-based therapy. All agents prescribed or inpatient treatments given within the first 3 months of starting a LOT were considered part of combination therapy. Any new agent prescribed/applied more than 3 months after initiating the LOT was defined as the start of a new LOT. A LOT was assumed discontinued at the start of a new LOT or in case of a gap between the prescriptions/administrations of the drugs belonging to the respective LOT of more than 3 months. If the same agent was prescribed/administrated again after a gap of more than 3 months (“re-start”), this was also defined as a new LOT. The 3-month gap definition was supported by sensitivity analyses where smaller and larger time-gaps of at least 1 month and at least 6 months between the start of platinum-based treatment and the next LOT were assessed. The 3-month gap definition also reflects the mean duration of cycle length and frequency of different commonly used anticancer interventions .HCRU was investigated in terms of the number of outpatient visits , the number of inpatient visits, and the number of days in the hospital. Both all-cause and EC-related utilization were assessed. For outpatient physician visits, cases were considered EC-related if any diagnosis was coded as EC . Hospitalizations were defined to be EC-related if the associated main diagnosis was EC.The HCRU items were assessed for the fixed 12-month pre-index period (baseline), the first 12 months after index, and the entire patient-individual follow-up period after the start of the post-platinum-based therapy. Generally, all items were reported per observed patient-year. In addition, the frequency of patients with at least 1 visit/hospitalization and the mean length of stay of all observed hospitalizations were reported.As the study addressed a retrospective anonymized dataset, no ethical review and no informed consent of patients were needed. However, the study protocol was reviewed by a scientific steering committee and the data owner. The work on the dataset conformed to all social security data protection requirements.Statistical analyses were performed using Microsoft SQL Server 2014 , STATA/MP 14 , and Microsoft Excel.Of the 6,832 identified patients with a valid EC diagnosis between January 01, 2011 and June 30, 2019, 1,469 had received anticancer treatment and, of those, 713 had received a platinum-based treatment. Out of these, 201 patients with post-platinum-based anticancer treatment , we identified at least one treatment before the platinum-based treatment Fig. : Four paAll 201 patients received a first platinum-based treatment in the baseline period. The most frequently observed first platinum-based treatment regimen was carboplatin and paclitaxel  in 90 patients (44.8%), followed by inpatient chemotherapy procedures without any specific information on prescribed chemotherapy agents in 86 patients (42.8%). Other carboplatin combinations were received by 14 patients (7.0%); cisplatin combinations were given to 10 patients (5.0%); one patient received oxaliplatin (0.5%)  and 22.9% had a radiotherapy (N = 46).Before start of the first post-platinum-based treatment, 13.4% of the observed patients had a surgery , and 36 patients received radiotherapy 17.9%). Of the 201 patients with a first post-platinum-based LOT, 79 patients (39.3%) received a second LOT. Three or more treatment lines were seen in 21 patients (10.4%). Kaplan–Meier estimation for the median time from first to second post-platinum LOT or death can be found in Supplemental Fig. 7.9%. Of The three most frequently observed anticancer agents in the first post-platinum-based LOT were carboplatin , paclitaxel , and doxorubicin . The treatment regimens varied from different inpatient chemotherapy procedures , endocrine therapy , and carboplatin combinations  to monotherapy options . An additional 65 combinations were identified, each being administered to fewer than seven patients . In total, 30 different agents and procedures in 45 different treatment regimens were seen in the second LOT , with 4.4 all-cause and 2.3 EC-related visits per observed patient-year Table . A gynecThe hospitalization rate in the analyzed patient sample was generally high. In the 12-month pre-index period, 183 patients (91.0%) had at least one inpatient stay. In total, 4.6 hospitalizations and 33.3 inpatient days per patient-year were observed in the baseline period, with 1.9/16.0 of these hospitalizations/inpatient days being related to EC. The number of hospitalizations per patient-year decreased to 3.3 (EC-related: 1.4) in the first 12 months of follow-up and increased to 5.2 (EC-related: 2.3) in the entire follow-up period. Correspondingly, the number of inpatient days per patient-year in the first 12 months after the start of post-platinum-based therapy was lower compared to baseline  but rose to 39.6 days per patient-year (EC-related: 17.2 days) in the entire follow-up period.The mean length of stay for an EC-related hospitalization was slightly longer than the average reported for all hospitalizations (8.5 vs 7.3 days) in the 12-month pre-index period, but marginally shorter when considering mean length of stay for all hospitalizations in the first 12 months after the start of the post-platinum-based therapy Supplementary file2 (PDF 201 KB)Below is the link to the electronic supplementary material.


Introduction: The present study had two objectives: 1) to investigate the effects of tumble turns on the development of inspiratory muscle fatigue (IMF) and compare this to whole swimming, and 2) to evaluate the effects of pre-induced IMF on the kinematic parameters of tumble turns. Fourteen young club-level swimmers (13 ± 2 years of ages) completed three swim trials.Methods: The first trial was used to determine the 400-m front crawl swim time at maximal effort (400FC). The other two trials consisted of a series of 15 tumble turns at the 400FC pace. In one of the turn-only trials, IMF was pre-induced (TURNS-IMF), whereas in the other turn-only trial it was not (TURNS-C).Results: Compared with baseline values, the values for maximal inspiratory mouth pressure (PImax) at the end of the swim were significantly lower at all trials. However, the magnitude of inspiratory muscle fatigue was less after TURNS-C (PImax decreased by 12%) than after 400FC (PImax decreased by 28%). The tumble turns were slower during 400FC than during TURNS-C and TURNS-IMF. In addition, compared to TURNS-C, turns in the TURNS-IMF were performed with higher rotation times and shorter apnea and swim-out times.Discussion: The results of the present study suggest that tumble turns put a strain on the inspiratory muscles and directly contribute to the IMF observed during 400FC swimming. Furthermore, pre-induced IMF resulted in significantly shorter apneas and slower rotations during tumble turns. IMF therefore has the potential to negatively affect overall swimming performance, and strategies should be sought to reduce its effects. The occurrence of inspiratory muscle fatigue (IMF) has been reported after 100-m , 200-m , 300 m, per se on the development of IMF. Moreover, it is also unknown how IMF affects turn performance. Regarding general swimming performance, it is known that pre-induced IMF increases metabolic acidosis during subsequent swimming, slows swimming speed, and increases stroke rate and breathing frequency  25-m swimming pool. The first trial was used to determine maximum-effort 400-m FC swimming (400FC) time. The remaining two trials consisted of a series of 15 tumble turns at the 400FC pace. We used the series of tumble turns to avoid the fatigue arising from the actual swimming component of the 400FC. The turns consisted of a 6-m swim into the wall, turn, and a 6-m swim from the wall. The rest period between each turn reflected the elapsed time that would have occurred had the swimmer been swimming 400-m FC at race pace. The 15 tumble turns reflect the number of tumble turns occurring during a 400-m swim in a 25-m pool. We defined the turn segment as swimming from the 5-m before the wall to the 5-m after the wall push-off . We seleFourteen young club level swimmers volunteered for this study  and forced expired volume in the first second of exhalation (FEV1) were recorded for descriptive purposes using a digital spirometer . Participants completed a minimum of three technically satisfactory manoeuvres with the highest recorded.PImax  and PEmax  were determined with the nose occluded and using a hand-held respiratory pressure meter whilst standing on poolside . Reliability was deemed present when the three highest manoeuvres from a series of manoeuvres were within 10 cmHThe participants completed three swimming trials. All of them were initiated from a push start, completed using the FC stroke, and occurred on separate days. The 400FC trial was started by pushing off the wall. In the sets of 15 turns, the swimmers started with the push-off from the bottom of the pool. An 800-m warm-up consisting of all swimming strokes was completed before each trial. The first trial was the 400FC. The remaining two trials, TURNS-C and TURNS-IMF, were administered in a counterbalanced order. During these trials participants were instructed to swim in and out of the turn at maximum speed from and to the 6-m mark. The appropriate rest period was given as described earlier. A researcher informed participants of the total time achieved for each turn and monitored the duration of rest during the series of tumble turns.2 to resting values after the cessation of hyperventilation  was used to induce IMF before the series of tumble turns in the TURNS-IMF trial. While standing and with the nose occluded, participants breathed through a flanged rubber mouthpiece connected to the inspiratory muscle trainer. The 1-way inspiratory value inside the trainer was set to open when participants produced 70% of their predetermined PImax (expiratory muscles were not loaded). A duty cycle of 0.60 was adopted  and an g regime . Particig regime . Particig regime , which ig regime . Due to g regime . Resultstilation . Therefo© 2006–2011, Joan Charmant & Contrib). Each trial was recorded by two cameras . The first underwater camera  was attached to a trolley at a depth of 0.36 m. The trolley was pulled by the operator at the same speed as the swimmer, with the head of the participant being the marker that the operator followed to control parallax  the turn, which started with the frame when the swimmer’s head reached the 5-m mark towards the turn wall and ended with the frame when the swimmer’s head reached the 5-m mark again after the turn; 2) the rotation, which started with the frame when the head and body started to lean down and ended with the frame when the feet touched the wall; 3) the apnea, which started with the frame that corresponded to the last breath before the turn and ended with the frame that corresponded to the first breath after it; and 4) the swim-out, which started with the frame when the toes were pulled back from the wall and ended with the frame when the ear broke the water surface. Two researchers performed video analysis to identify the initial and final frames of each analyzed phase. We used phase identification similar to that used in previous studies .PImax and PEmax were recorded pre- (baseline) and post- (end-swim) the 400FC and TURNS-C trials using a hand-held respiratory pressure meter . The values were used to assess IMF and expiratory muscle fatigue (EMF), respectively. In the case of the TURNS-IMF trial, baseline consisted of a pre-IMF inducement value (baseline) and a post-IMF inducement value (baseline after IMF inducement). Manoeuvres were made from a standing position on poolside (with the nose occluded) before and within 60 s following each trial. PImax was recorded from residual volume whereby participants were instructed to fully empty their lungs then inhale as hard and as fast as possible for approximately two to three s. PEmax was measured from total lung capacity and participants were instructed to fill up their lungs and exhale as hard and as fast as possible for two to three s. Before (baseline) and 1 minute after the end of each trial, blood samples were taken from the finger capillary to determine lactate ( to find a p-value of less than 0.05, assuming a power of 0.80 .The sample size was calculated considering a two-way repeated measures ANOVA with three interventions  and two repeated measurements (pre- and post-swim). The main endpoint was the PImax. This is the first study to examine the occurrence of a PImax decrease in response to a set of tumble turns. Therefore, our sample size calculation could only be based on previous studies of IMF in swimming. Swimming at 200-m race pace FC caused a significant PImax decrease ( of 0.72 . In the The magnitude of IMF and EMF were defined by the transient reduction in mouth pressure pre- (baseline PImax and PEmax) vs. end-swim, expressed as a percentage of the pre-test value. The turn parameters were averaged for the first seven turns and for the last seven turns and then used for the further analyses. This also permitted the change (∆) from the first to the last seven turns to be calculated.−], the turn parameters) as dependent variables. A one-way analysis of variance (ANOVA) was used to test for differences in IMF  between trials. When a significant interaction was detected (the F-ratio was significant), pairwise comparisons were performed with LSD . Moreover, the observed power and the effect size was calculated to estimate the variance between trials [partial eta squared: (η2)] and Cohen’s d for pairwise comparisons. The following guidelines were used to interpret the strength of η2 values . Normality of the distribution was assessed with the Shapiro-Wilk test, and sphericity was tested with the Mauchly test. A two-way analysis of variance (ANOVA) was performed using trials  and time  as factors and outcome measures , but no further reduction occurred in response to a series of 15 tumble turns . In comparison to baseline values (before IMF inducement for TURNS-IMF), PImax values at the end of swimming were significantly lower in all three trials . Large effect sizes were observed in all three trials . There was no significant difference in end-swim PImax between trials .All participants successfully completed all three trials. They finished tumble turns and swam-out before the 5-m mark. Baseline PImax was similar in all trials . In the p < 0.01, η2 = 0.30, observed power = 0.81)  and to TURNS-IMF (29% ± 17%) .There were significant differences in IMF between swim trials  . The mag2O in the TURN-C and 114 ± 25 cmH2O in the TURN-IMF; F = 1.71, p = 0.21, d = 0.4, observed power = 0.23). End-swim PEmax decreased to 91 ± 23 cmH2O only in the TURN-IMF . End-swim PEmax was lower  in the TURNS-IMF trial (91 ± 23 cmH2O) compared to the TURNS-C trial (105 ± 18 cmH2O).We measured PEmax only in the TURN-C and TURN-IMF. There was no significant difference between the trials in the baseline values of this parameter . In addition, the turns were slowest during 400FC . The turn durations in this trial were significantly longer compared to TURNS-C  and TURNS-IMF . However, there were no significant differences in total time durations between TURNS-C and TURNS-IMF.p < 0.01, η2 = 0.86. observed power = 1) . Apnea times were higher in the TURNS-C than in the 400FC  and in the TURNS-IMF . However, there were no significant differences in apnea durations between 400FC and TURNS-IMF (p > 0.05).Apnea duration during tumble turns shortened throughout the trials  . In addip = 0.01, η2 = 0.40, observed power = 0.78) , but not during the TURNS-C and TURNS-IMF. Moreover, there were significant differences in the duration of rotation between trials . Participants made the faster rotation during the TURNS-C compared to the TURNS-IMF  and the 400FC . In addition, rotation times were significantly longer in the 400FC than in the TURNS-IMF .The factor time interacted significantly with rotation time during tumble turns  . Rotatiop < 0.01; η2 = 0.76, observed power = 1) . Swim-out times were higher in the TURNS-C  than in the 400FC and TURNS-IMF .Swim-out time during tumble turns decreased throughout all trails  . In addip < 0.01, d = 1.8, observed power = 1, mean difference 5.7 mmol/l; 95% confidence interval 3.9–7.6), from 2.8 ± 1.2 to 4.7 ± 2.2 mmol/l in the TURNS-C , and from 2.7 ± 1.0 to 4.7 ± 2.6 mmol/l in the TURNS-IMF . There was significant difference between the trials in end-swim p < 0.01, η2 = 0.73, observed power = 0.99). This significant difference persisted between the 400FC and the TURNS-C  and between 400FC and TURNS-IMF . Heart rate also increased significantly during the 400FC, the TURNS-C, and the TURNS-IMF compared to baseline . Heart rates at the end of swim were not significantly different between trials .End-swim  . In the TURNS-C trial, the magnitude of IMF correlated moderately with ∆ apnea time , ∆ rotation time , and ∆ HR . Moreover, end-swim PImax correlated moderately with the rotation time  and the swim-out time . In the TURNS-IMF trial, end-swim PImax was moderately correlated with the swim-out  and rotation  times of the last seven turns. The magnitude of EMF was correlated strongly with Δ in rotation times during the TURNS-IMF . End-swim PEmax was correlated with apnea times of the first seven turns  and with the rotation times  of the last seven turns.No correlations were observed between the magnitude of IMF and turn parameters in the 400FC trial. However, end-swim PImax correlated strongly with the total turn time  the magnitude of IMF was less after a series of 15 tumble turns than after maximal 400-m FC swimming; and 3) pre-induced IMF decreased both apnea and swim-out times and increased rotation time during a series of tumble turns, but total turn duration was unaffected.To our knowledge, this is the first study to determine whether IMF occurred in response to a series of tumble turns and to investigate the effects of IMF on tumble turn kinematic parameters. The main findings were: 1) IMF occurred in response to tumble turns The magnitude of IMF observed after 400FC 28%;  was consBeside IMF after the maximal 400-m FC swim, this is the first time IMF has been demonstrated in response to tumble turns specifically. As expected, the PImax drop after the TURNS-C was smaller (PImax decreased by 12%) than after the 400FC (PImax decreased by 28%) . As both2 secondary to restricted breathing and maximal swimming could be another reason , as was the magnitude of EMF . This is not surprising given the requirement to flex the trunk during the rotation phase of the tumble turn, which is initiated by the upper fibres of the rectus abdominis and then maintained by the lower fibres of the rectus abdominis, with both obliques providing support , allows the swimmer to recognize the best moment to swim-out and to resume swimming with greater ease. Moreover, given that faster rotation is associated with better tumble turns, IMF therefore has the potential to negatively affect overall swimming performance, and strategies should be sought to reduce its impact.


Background: Emerging data indicate that the cellular microenvironment and interleukins (IL) play a crucial role in mycosis fungoides (MF). We aimed to explore the potential association between the composition of the cellular microenvironment and the expression of IL-22 and IL-17A in MF skin lesions.Methods: The study encompassed 16 cases of MF of different stages, for which sufficient skin tissue for immunohistochemistry and frozen tissue for reverse transcription-polymerase chain reaction, both taken from the same lesion, were available. Histological evaluation of eosinophils, neutrophils, CD20+, CD4+, CD8+, FOXP3+, CD56+, and CD1a+ cells was conducted. Additionally, mRNA expression levels of IL-22 and IL-17 mRNA were quantified using reverse transcription-quantitative polymerase chain reaction. SPSS version 28  was utilized for statistical analysis.Results: Among the cases examined, three were in the patch stage, eight in the plaque stage, and five in the transformation to high-grade large cell lymphoma (t-LCL). B-lymphocytes, neutrophils, and eosinophils were primarily observed in t-LCL cases. IL-22 levels displayed a significant association with IL-17A levels , FOXP3+ cells , and neutrophil density . No correlation was detected between IL-17A levels and the evaluated subtypes of microenvironmental cells.Conclusion: The microenvironment of MF lesions with t-LCL is noticeably different from early MF in terms of cellular composition. Histopathological identification of the cellular microenvironment may serve as an indicator of IL-22 tissue levels. These results implicate certain types of cells in IL-22 expression in the MF microenvironment and may contribute to advancing our knowledge on the pathogenesis and progression of the disease. Recent evidence suggests that the cellular microenvironment plays a critical role in the development and progression of solid tumors, as well as lymphomas, including cutaneous T-cell lymphomas (CTCLs) -4. It isApart from the cellular microenvironment, interleukins (IL) play a crucial role in MF, given their contribution to the establishment of an immunosuppressive and tumorigenic milieu . More rePart of this research work was previously presented as a meeting abstract at the 34th European Congress of Pathology (ECP 2022) on September 7, 2022.We retrospectively collected 30 MF cases of different disease stages. Hematoxylin and eosin (H&E)-stained sections were reviewed. Paraffin blocks were assessed for specimen adequacy. Finally, a total of 16 MF cases were included in our study. Inclusion criteria were diagnosis of MF, availability of sufficient fixed skin tissue and frozen tissue for reverse transcription-quantitative polymerase chain reaction (RT-qPCR), both taken from the same lesion, availability of clinicopathologic and follow-up data, and the provision of informed consent by the patients. We excluded patients who had a history of MF but were in complete remission.2 by using the program of Nikon Digital Sight DS-L2 Camera . Then, the number of eosinophils and neutrophils was counted in the total surface area, and their densities were calculated (number/mm2). A similar methodology was followed for the calculation of CD20+ B-cells and their density was evaluated on the corresponding slide. CD4+, CD8+, CD56+, and FOXP3+ cells were counted in relation to the total infiltrating population . A three-tiered scoring system for CD1a was performed , where the presence of dermal and epidermal CD1a+ cells was characterized as low if they were many but dispersed, intermediate if they tended to cluster, and high if they formed large groups. RT-qPCR was used to quantify tissue expression levels of IL-22 and IL-17 mRNA, as previously described [Three-μm successive paraffin sections were used for H&E stain and immunohistochemistry. Antibodies for CD20 , CD4 , CD8 , FOXP3 , CD56 , and CD1a  were used. Immunohistochemistry was performed using Bond Max  and Dako  automated immunostainers. On the H&E section of each case, the total surface area was measured in mmStatistical analysisAll data were examined for normal distribution with the Shapiro-Wilk test due to its higher power in small samples. The test provided evidence in favor of normal distribution (p > 0.05). After the assumption of normal distribution, the Pearson correlation procedure was used to examine statistically significant correlations among the continuously involved variables. For the needs of analysis, the IBM SPSS version 28  software package was used, and all tests were provided under a 5% level of significance.A total of 16 patients with MF were finally included in the study . Among them, 12 were male and four were female. Three were in the patch stage, eight in the plaque stage, and five in the t-LCL. Moreover, 11 patients had early-stage MF (IA-IIA) and five patients had advanced-stage MF (IIB-IV). Thirteen patients had not received any treatment prior to sample collection, two patients had undergone systematic treatment, and one had received a combination of skin-directed and systematic treatment.2, two of which were in t-LCL. No neutrophils were found in 10 cases (62.5%). B-cells were also absent in five cases, with the highest density (59.13 cells/mm2) being observed in a case with t-LCL. In most cases, the CD4:CD8 ratio was higher or equal to 4:1. Of note, the highest ratio (>20:1) was found in two t-LCL cases. Furthermore, the percentage of CD56+ cells was less than 5% in 10 cases. The percentage of FOXP3+ T-regulatory cells was <10% in seven cases, 10-25% in five cases, >25% in two cases, and non-evaluable in two cases, due to exhaustion of the lymphoma tissue. Finally, CD1a+ cells were scored as low in two cases, moderate in seven cases, and high in seven cases.Only three cases had more than 1 eosinophil/mmOverall, cases of patch and plaque stage MF displayed a remarkably different microenvironment in their cellular composition compared to MF lesions with t-LCL. Specifically, early MF lesions displayed no or few B-lymphocytes, eosinophils, and neutrophils, whereas these cells were abundant in t-LCL cases.A statistically significant association was observed between IL-22 expression levels and IL-17A levels . IL-22 tissue levels were also correlated with FOXP3+ T-regulatory cells  and with neutrophil density . Although the highest values of IL-22, neutrophil density, and FOXP3+ cells were recorded in t-LCL cases (Figure Little is known regarding the role of the cellular milieu and specific cytokines in MF development and evolution. In this study, a higher number of eosinophils, neutrophils, and B-lymphocytes was observed in t-LCL cases than in early-stage MF lesions. Regarding eosinophils, a significant correlation between the density of infiltrating eosinophils and the MF stage has been reported in the literature ,11. The In this study, IL-22 tissue levels were significantly associated with FOXP3+ Treg and neutrophil density. Recent research highlighted the potential involvement of Tregs in the regulation of IL-22 expression . ContrarStudy limitationsA limitation of this study is the small number of participants, which is why further multi-center studies involving a larger sample size are required to gain a deeper understanding of the role of the microenvironment in MF. Another limitation of the study is the absence of functional data regarding potential molecular and cellular mechanisms of interplay between different components of the MF microenvironment. Such information would provide additional substantiation for the current findings.The MF microenvironment shows diversity in cellular composition with different types of lymphocytes that are not morphologically distinguishable in everyday practice. IL-22 has not been well investigated in the MF microenvironment. The expression of IL-22 in the MF microenvironment seems to be regulated by FOXP3+ cells and leads to neutrophil recruitment. Although the cellular composition and cytokines cannot totally reflect the complexity of the immune milieu in MF, our results implicate IL-22 tissue levels in the cellular composition of the MF milieu that need further investigation. Delving into the MF microenvironment in terms of the cellular and cytokine composition has the potential to greatly enhance our understanding of the disease pathogenesis and, importantly, to facilitate the development of innovative targeted treatments.


Both the unmodified flour and the functionalized flour were employed to prepare water-based adhesives. For both flour systems, adding ACV at concentrations of at least 2.0% v/v enhanced the mold resistance of the adhesives and completely inhibited the development of A. niger mycelia for up to 90 days of storage. The adhesives made from the transesterified flour exhibited a higher shear strength for the paper bonding (ca. 8%) than the unmodified ones. Additionally, the ACV additive had no negative effects on the shear strengths of the water-based adhesives. All of the flour-based adhesives developed in this study had a higher shear strength for paper substrates than two locally available commercial water glues.As a byproduct of bromelain extraction procedures, pineapple stem flour is underutilized. Since water glues derived from gelatinization typically have poor mold resistance, this study aims to produce flour-based value-added products, such as mold-resistant water-based adhesives. To address this issue, this study explored the use of apple cider vinegar (ACV) as a low-cost, non-toxic, commercially available antifungal agent to improve the mold resistance of adhesives. Furthermore, laurate flour was produced via a transesterification of the flour and methyl laurate using a K Aspergillus niger, a black mold disease, usually contaminates food, especially sun-dried foods, grains, and nuts. Some strains of this fungus can also produce the mycotoxin ochratoxin A, which is responsible for human kidney damage. Additionally, the molds can also attack low-water-resistance adhesives. This is because many low-water-resistance adhesives can easily be penetrated by moisture, and the molds take advantage of this to grow on the surfaces of the adhesives [E. coli and S. aureus [Mentha pulegium and Lavandula angustifolia were mentioned to inhibit the growth of S. aureus in starch films [Cymbopogon citratus), neem oil, and oregano oils in starch-based films showed antifungal activity against Aspergillus niger (A. niger) [The global adhesives and sealants market is projected to reach approximately USD 85.8 billion by 2026  due to idhesives ,10. Jin . aureus . Combini. aureus . In addich films . Additio. niger) ,15.Vinegar is used in the food industry as a preservative by inhibiting fungal growth on vegetables due to its high content of organic acids, phenolic compounds, tannins, flavonoids, and carotenoids with antimicrobial activity ,17,18. TThis study aimed to assess apple cider vinegar’s in vitro antifungal properties on water-based adhesives made from pineapple stem flour because of its simplicity of use, abundance, low cost, and low toxicity. In order to test the viability of using apple cider vinegar as an antifungal reagent in polysaccharide-based adhesives rather than harmful synthetic chemicals, the shear strength of starch adhesives was measured and compared with that of other commercially available paper glues. The key findings from this work should promote the value creation of starch utilizations as natural raw materials for the development of biobased water glues for paper.2CO3; MERCK, Darmstadt, Germany), methyl laurate , and ethanol . Water  was used to prepare the water glue samples. Apple cider vinegar  was purchased from a local supermarket in Nakorn Pathom, Thailand.Hong Mao Biochemicals Co., Ltd.  supplied the pineapple stem flour (PAF), which is a by-product of bromelain extractions . Before further use, the flour was dried at 65 °C for 48 h. The commercially available analytical-grade chemicals that were used include dimethyl sulfoxide , potassium carbonate (KAspergillus niger TISTR3281 (or A. niger) was purchased from the TISTR Biodiversity Research Center, Pathum Thani, Thailand. Cardboard paper (100 g) was supplied by PT TRANG CO., LTD., Trang, Thailand. Two commercial water glues were purchased from local shops in Nakorn Pathom, Thailand.Potato dextrose agar  and potato dextrose broth  were also used for this study. 2CO3 (0.25 g) and methyl laurate (19.81 g) were added to the gelatinized mixture. The transesterification reaction between the PAF flour and methyl laurate underwent at 110 °C for 1 h. The reaction was subsequently cooled to room temperature. The resultant solid product was suspended in 200 mL of ethanol, agitated for 15 h, and then washed three times with ethanol. After filtering and drying in an air oven at 65 °C for 15 h, the esterified starch product (denoted as m-PAF) was obtained as a light-yellow powder. The molecular weight of the PAF and m-PAF samples were determined by gel permeation chromatography using Agilent Waters 2414, column: TSKgel G5000PW (300 × 7.5), in water as the solvent. The transesterification efficiency was measured in terms of the degree of substitution (DS) of the ester group on the starch backbone using a 1H NMR spectrometer (Bruker AVANCE 600 series FT NMR spectrometer (600 MHz)). The chemical shifts were reported in ppm (d6–DMSO at 2.50 ppm). The degree of substitution was determined as follows: 10 mg of m-PAF was added into a 1.5 mL screw-cap vial equipped with a magnetic stir bar. Around 1000 µL of deuterate solvent as d6–DMSO was added and stirred at 60 °C for 3 h. After that, the 700 µL mixture was transferred to an NMR tube. The signal was recorded as follows: 32 scans at 50 °C. The degree of substitution (DS) was determined by Equation (1) [I (0.85 ppm) refers to the integration of the proton signal corresponding to the terminal position of the laurate chain at C-12 and I (3.6–5.4 ppm) refers to the integration of the proton signal of the glucose unit.In a 500 mL round-bottom flask equipped with a magnetic stir bar, a suspension of the PAF flour (5 g) in 50 mL of DMSO was kept at 75 °C for 3 h to allow for effective starch gelatinization processes. Subsequently, Ktion (1) , which iv/v. A light-yellow water glue was obtained.One gram of either PAF or m-PAF and 20 mL of water were placed in a clear zipper polypropylene bag. The mixture was then manually agitated in the zipper bag, before being heated seven times in a household microwave oven  for one minute at a time. Then, apple cider vinegar (ACV) was added into the aforementioned flour mixture in proportions of 0.3, 0.625, 1.25, 2, 2.5, and 3% A. niger is a representative of the fungi family and is easily found in starchy foods. The fungal strain was incubated in a potato dextrose broth (PDB) at 35 °C for 7 days. The suspension formed by A. niger and spores was collected and transferred to a sterile tube. The optical density (OD) of the suspension was adjusted to 0.05 using a UV–visible spectrophotometer at 600 nm, adapted by Su et al. and Larregle et al. [e et al. ,25.A. niger fungus. First, 5 mL of the adhesive was mixed with 25 mL of the prepared aqueous PDA solution in a sterile tube, after which the mixture was placed in a sterile petri dish (13 mm in diameter). Sterile disks with a diameter 6 mm were soaked in the suspension of A. niger. To evaluate the antifungal activity of the ACV-containing water glues, the fungus-containing sterile disks were placed at the center of the agar medium mixed with the glue at varying ACV concentrations. The petri dishes were incubated at room temperature for up to 90 days. The fungal mycelial growth was monitored. The effect of the ACV concentrations in the PAF (or m-PAF) water-based adhesives on the fungal mycelial growth was calculated in terms of the percentage of mycelial growth inhibition (MGI) using the following Equation (2) [dc = the mean diameter (mm) of the fungal mycelial growth in the control treatment and dt = the mean diameter (mm) of the fungal mycelial growth in the sample-containing treatment.A mixture containing 39 g of mixed PDA powder in 1 L of RO water was sterilized in an autoclave  at 121 °C for 15 min. The previously prepared water glue samples were then used to monitor the mycelial development of the tion (2) :(2)MGI  was positioned at the end of each piece see . A paintp-values ≤ 0.05).The results were reported as a mean value and standard deviation (SD). The descriptive statistics were calculated using version 18 of the predictive analytics software (PASW) program . A one-wBased on the GPC and 1H NMR results, the properties of PAF and m-PAF are shown in 6 Da) [6 Da) [6 Da) [The molecular weight of PAF (106 Da) lies in the same range as other starches, including wheat starches (6.5 × 106 Da) , 51% of ) [6 Da) , and 80%) [6 Da) . The polA. niger in the water glues derived from the unmodified starch (PAF) and ACV/PAF mixtures under controlled conditions (room temperature). With moisture in the air, mold and bacteria easily attacked the adhesives produced from the unmodified starch. The round black shades in the figures reflect the growth of the fungi in the adhesive, while the white spots represent some bacterial colonies . Although smaller black shades of mold growth were observed with the increased ACV concentrations, as seen in v/v. v/v and above. In line with a previous report [v/v). It is possible that the improved antifungal activity of the ACV/PAF mixtures was due to the lower cellular pH (from 4.5 to 2.6). According to Kara et al., acidity is associated with antimicrobial activity. The antibacterial activity against S. aureus and E. coli (ATB: 57) was enhanced by the low pH of apple vinegars  [s report , adding nd Gala) .A. niger fungus can be seen as dark shades on the plate, and the white spots in the PAF and m-PAF samples are showing evidence of some bacterial growth. When 0.3% ACV is combined with the adhesives, the white spots disappear, implying inhibited bacterial growth. This result agrees well with previous work reported by Baysal et al. [L. monocytogenes and S. aureus) and Gram-negative  bacteria [Due to the high mold resistance of the water-based adhesives because of the optimum ACV concentrations, no fungal growth was observed for very long storage of up to 90 days see also . As a rel et al. . Due to bacteria .v/v ACV shows the highest shear strength for the paper substrate (v/v ACV are not significantly different (p ≤ 0.05). Nevertheless, the high shear strength values of 1.25–1.35 MPa for all adhesives studied suggested that the addition of ACV does not deteriorate the mechanical properties of the flour-based adhesive. Notably, all of the produced adhesives have significantly superior shear strengths to two commercially available water glues.The ACV/PAF adhesive with 3.0% ubstrate , while tv/v and below was observed based on the results in p ≤ 0.05). At an ACV concentration of 1.25% v/v, the mycelial growth inhibition improved moderately. In the examined ACV/m-PAF water-based adhesive, the 2.0% v/v ACV concentration effectively inhibits the growth of fungi and bacteria  in the m-PAF. By adding a small amount of ACV, the pH of the ACV/m-PAF adhesives is found to be much lower than that of the m-PAF one. The gradual decrease in pH of the ACV/m-PAF adhesives due to the increasing ACV concentrations occurred along with a significant improvement in the antifungal activity of the ACV/m-PAF adhesives. With an ACV concentration of 2.0% v/v or more, the ACV/m-PAF adhesives could completely stop the fungal and bacterial growths (as shown in As shown in p ≤ 0.05) were obtained for the ACV/m-PAF water-based adhesives at ACV concentrations of 2.0–3.0% v/v. Thus, the results suggest the feasibility of utilizing ACV as an effective antifungal agent in water glues, as it can extend the glues’ shelf life without worsening their mechanical properties.In comparison, the shear strengths of the papers obtained from the m-PAF-based adhesives are slightly higher (ca. 8%) than those of the PAF water glues . Notablyv/v to produce mold-resistant flour-based adhesives, it is important to note that the mycelial growth rate of these adhesives differs at ACV concentrations that are less than 2.0% v/v. Despite the small improvement in the mechanical properties of the esterified flour adhesive compared to the adhesive produced from the unmodified flour, it is not entirely understood whether the ester groups functionalized on the polysaccharide backbone play an important role in the antifungal activity of flour-based adhesives. From v/v, the high pH of the gelatinized ACV/m-PAF may be the major cause of the somewhat quicker mycelial development and lower mold resistance in the ACV/m-PAF. On the other hand, at increased ACV concentrations, the mycelial development rates were found to be quite similar for both the ACV/PAF and ACV/m-PAF cases. Hence, adding a suitable amount of ACV to flour-based adhesives could be more effective in enhancing antifungal activity than using modified flour as an adhesive raw material. As previously reported [A. flavus at aw 0.98 and pH 5.5 compared to pH 3.0 [A. niger mycelia in the medium having a pH of around 7.0 [A. niger. This growth inhibition was concentration-dependent, with the greater acid concentrations having the greatest impact.To better understand the effect of ACV in suppressing mycelial growth in the investigated adhesives, the mycelial diameters in the PAF and m-PAF systems were compared in reported , pH playo pH 3.0  and the ound 7.0  support ound 7.0 ), and poound 7.0 . The aciound 7.0 . Diffusiound 7.0 ,41,42,43ound 7.0 . The pheound 7.0 . The resound 7.0 , such thv/v, the salts in the modified flour-based adhesives inhibit fungal growth more effectively than the salt-free adhesives produced from PAF. Previous works have demonstrated the antifungal properties of ACV; however, this is the first study to investigate the use of ACV in adhesive applications. In the m-PAF adhesives, the addition of ACV caused the formation of salts  due to acid–base neutralization between the basic gelatinized m-PAF solution (pH 10.2) and the acids in the ACV. Salinity effects have been reported to inhibit fungal mycelial development ,45. The v/v to flour-based adhesives, it is possible to obtain long-shelf-life water glues with high antifungal activity against A. niger, a common fungus found in starchy foods. The chemical composition (organic acids and polyphenols) and pH of ACV should play an important role in the satisfactory antifungal activity of the ACV-containing adhesives. Additionally, the bonding strength of the adhesives on the papers suggested that ester functionalization slightly improved the mechanical properties of the flour adhesives. The results in this work indicated that pineapple stem flour, a low-cost, abundant, and underutilized source of polysaccharides, is applicable as a biobased raw material for the production of eco-friendly water-based adhesives that give high bonding strengths to paper substrates and are superior to two commercially available water glues. The key findings of this study support the viability of producing biobased adhesives with a cheap, non-toxic, and naturally based antifungal agent, which serves well from the perspective of health-conscious customers. Therefore, the novelty of this work should serve as a useful resource for further research and development of additional low-cost, eco-friendly, biobased products in adhesive and other applications.This study has demonstrated that apple cider vinegar (ACV) has great potential as an antifungal agent, significantly enhancing the fungal resistance of water-based adhesives made from pineapple stem flour (PAF) and esterified PAF (modified PAF or m-PAF). By adding ACV at a low concentration of 2.0%


Chylous abdominal effusions are serious complications that can be triggered by various aetiologies. The biochemical diagnosis of chyle leakage in ascites or in peritoneal fluid capsules relies on the detection of chylomicrons. Assaying the fluid’s concentration of triglycerides is still the first-line tool. Given that only one comparative study has sought to quantify the value of the triglyceride assay for diagnosing chylous ascites in humans, our objective was to provide practical triglyceride thresholds.We conducted a 9-year, retrospective, single-centre study of adult patients and compared a triglyceride assay with lipoprotein gel electrophoresis for the analysis of 90 non-recurring abdominal effusions  of which 65 were chylous.A triglyceride threshold of 0.4 mmol/L was associated with a sensitivity > 95%, and a threshold of 2.4 mmol/L was associated with a specificity > 95%. According to Youden index, the best threshold was 0.65 mmol/L with a sensitivity of 88 (77-95)%, a specificity of 72 (51-88)%, and, in our series, a positive predictive value of 89 (79-95)% and a negative predictive value of 69 (48-86)%.In our series, cut-off of 0.4 mmol/L could be used for ruling-out diagnosis of chylous effusions, while cut-off of 2.4 mmol/L could be used for reasonably confirming diagnosis. The peritoneal fluid  is located between the multifunctional, structurally complex mesothelial layers of the body  has various congenital or acquired causes: cancer, inflammation, cirrhosis, or trauma  (the chylomicron’s main constituent) is still the first-line, rapid, biochemical test for chylous ascites   were sent to the laboratory. In accordance with the French legislation on non-interventional studies of routine clinical practice, the patients were not required to give specific consent but were free to refuse the use of their medical data. All collected data were anonymized before analysis.To ensure that all samples were independent and to avoid giving too much statistical weight to particular patients, we excluded recurring or multiple samples. Samples whose origin was unclear were also excluded. Of the 90 samples included , 65 (72%) were considered to be chylous (on the basis of the electrophoresis results) and thus confirmed the initial clinical suspicions.Triglyceride and cholesterol concentrations in abdominal liquids were measured in an automated manner by combining three enzyme reactions with a final Trinder reaction and colorimetric detection. Triglycerides are first hydrolysed by lipoprotein lipase. Two auxiliary enzymatic reactions (mediated by a peroxidase) give a detectable quinone-imine. The TG assays were performed with a DiaSys kit  on a Konelab 30i system  up until March 2018 (65 samples) and with a TRIGL kit on a Cobas 8000 system  thereafter (25 samples). The TG assay gave linear results from 0.0 to 11.3 mmol/L (for DiaSys) and from 0.1 to 10.0 mmol/L (for Roche).After centrifugation (4437xg for 10 min), the fluid’s appearance was assessed both macroscopically and by measuring the absorbance at 620 nm (Konelab 30i), with the following criteria: 0.001 - 0.080: clear; 0.081 - 0.800: opalescent; 0.800 - 1.000: milky. Lipoproteins were separated electrophoretically with a validated, manual, semiquantitative technique (based on the Hydragel Lipo + Lp(a) K20 kit, Sebia, France), stained with Fat Red reagent (Sudan Red 7B-Aldrich) and revealed with a Hyrys2 system (Sebia)  curve and its 95% confidence interval (95%CI), calculated with a bootstrap method. For a given threshold, the sensitivity, specificity, positive predictive value, and negative predictive value were quoted with their exact 95%CIs . “Best” threshold was defined as the threshold that maximizes the Youden index, and its confidence interval obtained by bootstrap.Since continuous variables studied here were not expected to have a Gaussian distribution , comparison between chylous and non-chylous were preplanned to be made using non-parametric methods (Wilcoxon-Mann-Whitney). This avoids both multiple testing issues and unknown control of global Type-I error due to hierarchical testing.vs. non-chylous samples were compared using Fisher’s exact test  or the Wilcoxon-Mann-Whitney test (for quantitative data). Since the analysis was descriptive, P-values were not corrected for multiple testing. All statistical analyses were performed using R software (version 4.0.2) and the pROC package (version 1.18.0) : 0.79 (0.68-0.90) did not provide additional diagnostic value and might even decrease it.As shown in et al. In contrast, our threshold for good specificity (> 95%) was quite close to Thaler et al.’s value. Although the two studies had similar diagnostic objectives, the respective datasets probably differed in several respects. With regard to analytical variables, we do not know whether the respective assay methods were correlated. One can also hypothesize that a low proportion of the electrophoresis results could be false positives , aetiologies, and prescribing habits (e.g. prescription of the electrophoresis assay). The presence of inter-study differences in TG thresholds suggests that second-line techniques (such as gel lipoprotein electrophoresis or the observation of stained samples under the microscope) should be used more frequently. Relative to our study, Thaler et al. used the same R package and a similar statistical approach but did not report a CI for the proposed threshold. Our 95%CI is broad and includes the threshold value that Thaler et al. determined from the Youden index. The inter-study difference might be not significant and might reflect inter-individual variability and the small size of both datasets.To the best of our knowledge, there is only one comparative study in the indexed medical literature on the TG assay’s value in diagnosing chylous ascites in humans : 2.1 mmol/L when maximizing Youden index (with a sensitivity of 87% and a specificity of 89%), 1.7 mmol/L for a sensitivity greater than 95%, and 2.8 mmol/L for a specificity greater than 95% , the time that the sample had spent at room temperature prior to the assay (which can affect the TG stability), or the patient’s nutritional status (which can influence chylomicron generation). Fifthly, we did not distinguish between the various aetiologies of chylous ascites and abdominal chyle leakage. Sixthly, recruitment bias might have affected the “control” fluid samples, e.g. apolipoprotein apoB48 (for research purpose), could be interesting (Analysis of a more direct biomarker of chylomicrons,


Alzheimer's disease (AD) dementia is the most frequent cause of neurodegenerative dementia. The cognitive and behavioral symptoms associated with this disorder often have overlapping characteristics, potentially resulting in delayed diagnosis or misdiagnosis. This study aimed to assess the level of peripheral blood neurofilament light chain (NfL) and total tau (t-tau) protein in AD patients and investigate their relationship with cognitive impairment. The study included 80 participants of both sexes between the ages of 60 to 85 years. The participants were divided into two groups, consisting of 40 individuals in the control group (mean age 75±6.6 years) who had no cognitive or functional impairments and 40 AD patients (mean age 74.98±5.03 years). This study utilized the DSM-5 diagnostic criteria for major or mild neurocognitive disorder attributed to Alzheimer's disease (AD). The clinical and biochemical features of all participants were documented, and the Alzheimer's disease Assessment Scale cognitive subscale (ADAS-cog) scores were evaluated. Sandwich ELISA was employed to determine serum NfL and t-tau protein levels. The median serum NfL and t-tau protein levels in AD patients were significantly higher than those of the controls . Age was positively correlated with NfL, t-tau levels, and ADAS-cog. Although elevated NfL and t-tau protein levels may play a role in disease progression, their diagnostic value for AD was limited. Plasma neurofilament light (NFL) chain and total Tau (t-tau) proteins have emerged as two potential biomarkers for the detection of Alzheimer's disease (AD) dementia. T-tau levels increase in pathology-confirmed AD and are recognized as a neurodegeneration biomarker in contemporary AD dementia research frameworks . On the Research has indicated that blood t-tau has limited diagnostic potential for AD , whereasLimited data is available regarding the impact of critical biological factors, such as age and sex, on plasma NfL and t-tau levels . The objAn observational case-control study was carried out at the Ibn-Rushed Psychiatric Teaching Hospital, Baghdad, including 80 Iraqi participants of both sexes aged between 60 to 85 years. The participants were divided into two groups: 40 patients with clinically diagnosed AD (mean age 74.98±5.03 years) referred by a psychiatrist blinded to the biomarker results with no other psychiatric disorders, and a control group consisting of individuals without any psychiatric disorders.The DSM-5 diagnostic criteria for major or mild neurocognitive disorders attributed to AD dementia was used. Patients had a positive family history of AD dementia with clear evidence of a decline in memory and learning based on detailed history and neurocognitive assessment by the Mini-Mental State Examination. In addition, the patients were well-established cases and had been visiting the hospital regularly. Furthermore, AD dementia patients had no evidence of mixed etiology .The other group represented the healthy control group, comprising 40 age- and sex-matched subjects (mean age 75±6.6 years). They were functionally intact with no cognitive impairment on the Mini-Mental State Examination. Individuals with chronic diseases, such as autoimmune and neurodegenerative disorders, were excluded from the study.All the participants in the study were subjected to the same study protocol and assessed by a psychiatrist with experience in administering the Alzheimer's Disease Assessment Scale - Cognitive Subscale (ADAS-cog) to evaluate cognitive function. The version used in this study was the basic ADAS-Cog with 11 items. This test takes about 30 minutes and is scored from 0 to 70: higher scores indicate greater cognitive impairment (CI). The main areas of the cognitive domains evaluated are memory (50%), language (28%), praxis (14%), and command understanding (8%). A high score indicates poor performance .Three milliliters of venous blood samples were collected in gel tubes in the morning, after 12-14 hours of fasting, from all participants by a certified laboratory technician. The samples were centrifuged at 3000 rpm for 10 minutes, and the resulting sera were frozen at -20°C until further processing. For the quantitative measurement of NfL and the t-tau protein, an enzyme-linked immunosorbent assay (ELISA) was used (Demeditec Diagnostics/Germany). The microtiter plate reader was set to read at a wavelength of 450 nm.The statistical analyses were conducted utilizing SPSS software version 25.0 . Continuous data were evaluated for normality using the Shapiro-Wilk test. Data that exhibited normal distribution were presented as the mean and standard deviation and were analyzed using an independent t-test. For data that were not normally distributed, a natural log transformation was applied and then converted to Z-scores, after which an independent t-test was performed. Categorical variables were presented as numbers and percentages and analyzed with the Chi-square test. To assess the discriminative ability of NfL and t-tau proteins in distinguishing AD from controls, the receiver operating characteristic curve (ROC) was utilized. A combination of both biomarkers was used in the logistic regression model. The cut-off value with the highest sensitivity and specificity was calculated according to Youden Index . The cor2, significantly lower than the mean BMI of controls (26.14±3.37 kg/m2) with a highly significant difference. In addition, patients with AD exhibited a significantly higher ADAS-cog score than controls (48.73±7.8 vs. 4.95±1.61), with a highly significant difference . Similarly, the sex distributions of the two groups were comparable, although females were more numerous among patients. The mean BMI of patients with AD was 21.12±2.65 kg/mfference .The mean Z-score of the natural log of serum level of t-tau protein in patients with AD dementia was 0.35±0.11, significantly higher than that of controls (-0.24±0.15) with a highly significant difference. Similarly, patients with AD dementia showed a significantly higher mean Z-score of the natural log of NfL (0.26±0.14) than controls (-0.18±0.09) with a significant difference .The AUC for t-tau protein was 0.693 . At a cut-off value of t-tau= 20.61 pg/ml, the sensitivity and specificity of the test were 0.68 and 0.57, respectively. The AUC for NfL was 0.664 . At a cut-off value of NfL= 11.21 pg/ml, the sensitivity and specificity of the test were 0.68 and 0.52, respectively .Combining the two markers increased the AUC . The sensitivity and specificity of the test at the above cut-off values were 75% and 60%, respectively .We used Pearson's correlation to investigate the relationship between NfL, t-tau, and ADAS-cog with age and BMI in the patient group. T-tau protein showed a significant positive correlation with age , NfL , and ADAS-cog score . NfL correlated positively with age  and ADAS-cog score . The ADAS-Cog score correlated positively with age  .The present study aimed to evaluate serum NfL and t-tau proteins in individuals diagnosed with AD dementia and determine their relationship with ADAS-cog. The study revealed a significantly higher ADAS-Cog score for patients with AD dementia compared to controls, suggesting that AD patients have moderate to severe cognitive impairment (CI) . This reMoreover, patients with AD dementia had considerably greater serum t-tau protein levels than controls, consistent with the results of numerous international studies -19. IncrAccording to the current study, there was a positive correlation between the ADAS-Cog score and serum t-tau level, consistent with the results of a previous study conducted in the United States . A recenConversely, a meta-analysis reported that the levels of NfL in both blood and CSF were unable to differentiate between AD dementia and other conditions that are similar to it, such as vascular dementia, dementia with Lewy bodies, Parkinson's disease dementia, idiopathic normal pressure hydrocephalus, and posterior cortical atrophy . ElevateTo summarize, the study demonstrated that while serum NfL and t-tau protein levels were higher in AD patients, they exhibited poor diagnostic performance in distinguishing AD from healthy controls. The study also found that age was significantly associated with the occurrence of AD biomarkers, including NfL, t-tau protein, and ADAS-Cog scores, suggesting that advanced age is a contributing factor to the increased prevalence of AD biomarkers. These results have important implications for the diagnosis and management of AD and underscore the need for more reliable and sensitive diagnostic markers for this disease.